Electrochemical methods and devices for amending urine samples for immunosensor detection

ABSTRACT

The present invention is directed to methods and devices for amending undiluted and partially diluted urine samples in a manner suitable for performing immunoassays for target analytes, for example NGAL. Generally, the urine sample is treated with reagents including at least one of buffer materials, water soluble proteins, urease, and other interferent mitigants. These reagents control the pH of the urine sample in a manner suitable for immuno-binding reactions and ameliorate interferences, particularly during the detection step.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent applicationSer. No. 14/206,532 filed on Mar. 12, 2014, which claims priority toU.S. Provisional Application No. 61/781,576 filed on Mar. 14, 2013, theentireties of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to analytical testing devices and methods forperforming electrochemical immunoassays. Specifically, the inventionrelates to analytical testing devices and methods for performingelectrochemical immunoassays on urine samples, and in particular usingpoint of care immunoassays for testing for analytes in urine samples.

BACKGROUND OF THE INVENTION

Clinical analyses are generally performed in blood or derivates thereof(e.g., serum and plasma). Additionally, some clinical analyses areperformed in urine, for example home pregnancy tests for human chorionicgonadotropin (hCG). However, urine typically comprises urea, which issynthesized as part of the urea cycle as a vehicle for the excretion ofexcess nitrogen. Urea is a known denaturant of proteins (e.g., enzymes),which are conventionally used in clinical analyses such as enzyme-linkedimmunosorbent assays (ELISA). Consequently, urine comprising ureagenerally presents a problem for clinical analyses (i.e., urea may causethe denaturation of the enzymes used in the clinical analyses), andthere are currently no known solutions to overcome the potentialdenaturation of the proteins used in the clinical analyses.

Nonetheless, as clinical analyses evolve there is a desire to expand onclinical analyses performed in urine. For example, historically, amarker such as serum creatinine has been used to diagnose acute kidneyinjury (AKI). However, serum creatinine measurements may be influencedby muscle mass, muscle metabolism, gender, race, hydration status andmedications. Additionally, the delay (e.g., up to 2-3 days after injury)and unreliability in serum creatinine rise may result in delayeddiagnosis, which can translate to irreversible kidney damage prior totreatment. Therefore, the disadvantages of measuring serum creatininefor clinical purposes have necessitated the identification of novelearly kidney injury markers. One such marker is neutrophilgelatinase-associated lipocalin (NGAL), which may be present in urineand/or plasma.

NGAL was identified as a secreted protein from granules of activatedhuman neutrophils. See Devarajan, “Review: Neutrophilgelatinase-associated lipocalin: A troponin-like biomarker for humanacute kidney injury,” Nephrology 15 (2010) 419-428. Specifically, NGALis a 25-kDa lipocalin that exists in monomeric and homo- andheterodimeric forms, the latter as a 46-kDa dimer with human neutrophilgelatinase. Lipocalins possess many different functions, such as thebinding and transport of small hydrophobic molecules, nutrienttransport, cell growth regulation, and modulation of the immuneresponse, inflammation, and prostaglandin synthesis. Specifically, theNGAL protein is believed to bind small lipophilic substances such asbacteria-derived lipopolysaccharides and formylpeptides, and mayfunction as a modulator of inflammation.

Renal injuries or disease, such as AKI, can result from a variety ofdifferent causes (such as illness, acute injury, sepsis, andradiocontrast nephropathy). NGAL may be utilized as an early marker foridentifying AKI, as it is produced by nephrons and renal tubular cellsin response to different types of injury in both animal and humanmodels. Specifically, it has been proposed that NGAL plays an importantrole in renal protection, regeneration, and repair. For example, NGALlevels rise in acute tubular necrosis from ischemia or nephrotoxicity,even after mild “subclinical” renal ischemia, as compared to normalserum creatinine levels, which further substantiate the recognition ofNGAL as an early renal injury marker. Moreover, NGAL is known to beexpressed by the kidney in cases of chronic kidney disease and this issuggested to be predictive of disease stage. It has also been suggestedthat the degree of NGAL expression may distinguish amongst AKI, prerenalazotemia, and chronic kidney disease. Additionally, NGAL has beensuccessful in predicting clinical outcomes in several common clinicalscenarios. For example, in the future, NGAL may be used to expedite thedrug development process or perhaps act as a safety marker duringclinical trials of potentially nephrotoxic agents.

NGAL is rapidly secreted into the urine, where it can be easily detectedand measured, and precedes the appearance of other known urinary orserum markers of ischemic injury. The protein is resistant to proteases,suggesting that it can be recovered in the urine as a faithful marker oftubule expression of NGAL. Further, NGAL derived from outside of thekidney, for example, filtered from the blood, does not appear in theurine, but rather is quantitatively taken up by the proximal tubule.

A variety of immunoassays are known in the art for detecting NGAL. Forexample, WO2010058378A1 outlines the immunoassay measurement of NGAL todiagnose AKI and the like and reports on the relative amounts ofmonomeric, dimeric, and heterodimeric NGAL to more accurately reflectdisease. Further, Antibody Shop A/S describes a kit and components forthe detection of NGAL (WO2006066587A1). United States Patent ApplicationNo. 2010/0227775 ('775) (Birkenmeyer et al. entitled: Immunoassays andkits for the detection of NGAL) discloses NGAL immunoassay methods andkits in which samples, e.g., blood, plasma, serum, and urine, suspectedof containing human NGAL monomer and human NGAL dimer are contacted withat least one first antibody (e.g., a capture antibody) to form a firstantibody/human NGAL complex. The at least one capture first antibodybinds to human NGAL and is an antibody (e.g., a capture antibody)selected from the group consisting of an antibody produced by murinehybridoma cell line 1-2322-455 having ATCC Accession No. PTA-8024 and anantibody produced by murine hybridoma cell line 1-903-430 having ATCCAccession No. PTA-8026. Additionally, United States Patent ApplicationNo. 2009/0176274 discloses a recombinant human NGAL (rhNGAL) that can beemployed as calibrator or control in an NGAL immunoassay. Determiningthe concentration of NGAL antigen in a test sample can be adapted to avariety of automated and semi-automated systems (including those whereinthe solid phase comprises a microparticle), as described, e.g., in U.S.Pat. Nos. 5,089,424 and 5,006,309, and as commercially marketed, e.g.,by Abbott Laboratories (Abbott Park, Ill.) as ARCHITECT®.

While troponin assays (cardiac troponin I (cTnI) and cardiac troponin T(cTnT)) are not performed in urine in standard clinical analysis, atleast one study has investigated these tests in urine, albeit withassays not specifically formatted for this sample type. See, e.g.,Zeibig et al., Renal elimination of troponin T and troponin I: ClinicalChemistry 49, 1191-3, 2003.

The '775 application also discloses that these assays, kits, and kitcomponents can be employed in other formats, for example, onelectrochemical or other hand-held or point-of-care assay systems, e.g.,The present disclosure is, for example, applicable to the commercialAbbott Point of Care (i-STAT®, Abbott Laboratories) electrochemicalimmunoassay system that performs immunoassays. Immunosensors and theirmethods of manufacture and operation in single-use test devices aredescribed, for example in, U.S. Pat. No. 5,063,081, U.S. PatentApplication Publication No. 2003/0170881, U.S. Patent ApplicationPublication No. 2004/0018577, U.S. Patent Application Publication No.2005/0054078, and U.S. Patent Application Publication No. 2006/0160164,which are incorporated in their entireties by reference for theirteachings regarding same.

The i-STAT® immunoassay platform employs calf intestinal alkalinephosphatase [3.1.3.1] on the detection antibody in order to convert asubstrate (p-nitrophenylphosphate) into an electrogenic species(p-nitrophenol), detectable on an amperometric biosensor. The pH optimumfor the ALP reaction is 9.1. Using blood as the biological test specimenwith ALP does not impose a residual matrix effect on the system.However, using urine may be problematic especially consideringinterfering elements like urea (average 0.4M, pH 4-5), pH (range4.5-8.5; Jung et al., describe instability of ALP due to pH extreme andother characteristics of urine, Clinica Chimica Acta 131, 185-91, 1983;they also suggest to measure the pH of the test reaction and use thisinformation during ALP activity calculations) and electroactive species(B vitamins, ascorbic acid etc.). Specifically, these elements canreduce enzymatic activity as well as increase background currentgenerated during oxidation of species during analysis in the i-STAT®platform. As the current i-STAT® immunoassay cartridge in someembodiments may not include a full wash step after antigen is captured(instead the cartridge may be configured to perform a limited washstep), the ALP reaction is only partially cleared of potentiallyinterfering elements from the original urine sample. As urea is commonlyused as a biomolecular denaturant, it is anticipated that an enzyme suchas ALP may be denatured in the presence of urine in the i-STAT®cartridge. Indeed, urea inhibition of ALP activity is well known and isused as a means of differentiating ALP isoenzymes (Bahr et al., ClinicaChimica Acta 17, 367-70, 1967).

The mechanism of inhibition is believed to be through a noncompetitivepathway, up to a threshold of urea concentration (Rajagopalan et al, JBC236, 1059-65, 1961). Past this reversible inhibitive concentration, ureathen becomes an irreversible denaturant (Birkett et al., Arch. Biochem.Biophys. 121, 470-9, 1967). ALPs from different tissue sources have beenshown to have different susceptibility to the effect of urea, with theplacental enzyme being the most resistant and the bone-derived versionbeing the most sensitive (Birkett et al, 1967; Gorman and Statland,Clin. Biochem. 10, 171-4, 1977). Interestingly, Metz et al. (ClinicaChimica Acta 30, 325-30, 1970) showed that the inhibitory action of ureaon ALP activity was markedly increased by pre-incubation with urease,and the effect was enhanced by prolongation of the pre-incubationperiod. Metz proposed that this effect was due to an increase inammonium salts, especially at the reaction pH of 9.3 (ALP optimum pH9.1). Further, production of ammonium leads to an increase in pH,potentially compounding the effect (Dawson, R. M. C., Elliott, D. C.,Elliott, W. H. and Jones, K. M. (1986) Data for Biochemical Research,3rd Edn., Clarendon Press, Oxford, p. 555). No further adjustment of thereaction to reverse the effect of the urease enhancement of ureainhibition on ALP was attempted.

U.S. Pat. No. 6,824,985 teaches the use of excess urea (>225 mM urea) inurine-based immunochromatographic strip and plate assays in order toreduce or eliminate bias in the test due to varying urea concentrationsbetween biological samples. Another group employing an optical system,which monitors turbidity due to agglutination, teaches that urease canbe added to the immunoassay to ensure antibody/antigen aggregation isgenerated independent of the concentration of urea (see, e.g., U.S. Pat.No. 7,960,132). However, neither of these teachings could be applied tothe i-STAT® cartridge since a variable amount of urea may remain duringthe analysis cycle (limited wash after antigen capture) and theproduction of ammonium ions and increased pH, upon addition of ureasewould have to be addressed in order to prevent further inhibition of theALP reaction.

One other consideration for carrying out an immunoassay in urine is thereduced ability of urine to naturally act as an immunoassay blocker (toreduce background) due to the low level of protein (0-8 mg/dL) comparedto blood (6.3-8.2 g/dL). Additionally, the i-STAT® system has a limitedcapability of pretreating and conditioning the sample (e.g., use ofbuffers to adjust pH) as well as washing during the assay cycle in waysakin to the ARCHITECT® system (see, e.g., U.S. Patent ApplicationPublication No. 2011/028562)

Based on the foregoing, there remains a need for pretreating or suitablyamending substantially undiluted urine samples in a manner to reduceinterferences and ensure immuno-binding reactions occur reliably, formeasurements of various markers including NGAL and others, such as,Chlamydia, Legionella, various infectious disease agents, and variousdrugs of abuse (DOA). The present disclosure seeks to provide methods ofpretreating or suitably amending urine samples and reaction steps inways that are amenable to immunoassays using a device or system, e.g.,the i-STAT® system. As well, other objects, advantages and inventivefeatures, will become apparent form the detailed description providedherein.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is directed to a deviceconfigured to perform an immunoassay for a target analyte in a urinesample. The device includes a first region comprising reagentsconfigured to amend the urine sample. The reagents may comprise a watersoluble protein and a buffer. The device further includes a secondregion comprising at least one electrode configured to determine aconcentration of the target analyte in the amended urine sample. Thefirst region is configured to provide a dissolved concentration of thewater soluble protein within the amended urine sample in a range ofabout 1 to 225 mg/mL.

In some aspects, the immunoassay may be selected from the groupconsisting of a one-step immunoassay, a low wash immunoassay, and ahomogenous immunoassay. In some embodiments, the water soluble proteinmay be recombinant or non-recombinant human serum albumin, porcine serumalbumin, castor bean albumin, salmon serum albumin, bovine serumalbumin, or a mixture thereof.

In some embodiments, the buffer may be configured to adjust a pH of theurine sample to within a preselected range. The buffer may be selectedfrom the group consisting of: glycine, 3-(N-morpholino)propanesulfonicacid (MOPS), tris(hydroxymethyl)aminomethane (Tris), tricine, acetate,borate, 2-(N-morpholino)ethanesulfonic acid (MES), and2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES).

In some embodiments, the reagents may further comprise a scavengerconfigured to reduce non-specific current generation from electroactivespecies. The scavenger may be a vitamin B scavenger.

In some embodiments, the device may further comprise a componentconfigured to dilute the urine sample with a diluent. The diluted urinesample may comprise about 10% diluent.

In some aspects, the target analyte may be NGAL. In some additional oralternative embodiments, the target analyte may be selected from thegroup consisting of human chorionic gonadotrophin, troponin I, troponinT, Chlamydia, Legionella, acetaminophen, amphetamines, methamphetamines,barbiturates, benzodiazepines, cocaine, methadone, opiates,phencyclidine, marijuana, and tricyclic antidepressants.

In some embodiments, the reagents may further comprise urease configuredto reduce a urea concentration of the urine sample below a preselectedurea threshold. In additional embodiments, the reagents may furthercomprise glutamine synthetase or any other urea cycle enzyme configuredto consume ammonium. In additional embodiments, the reagents may furthercomprise a sequestering enzyme configured to reduce and sequester excessphosphate below a preselected phosphate threshold.

In yet another embodiment, the present invention is directed to a methodfor performing an immunoassay for a target analyte in a urine sample.The method comprises providing a test device with reagents disposed in afirst region of the test device and at least one electrode disposed in asecond region of the test device. The reagents comprise a water solubleprotein and a buffer. The method further comprises amending a urinesample with the reagents such that a dissolved concentration of thewater soluble protein within the amended urine sample is in a range ofabout 1 to 225 mg/mL. The method further comprises determining aconcentration of the target analyte within the amended urine sampleusing the at least one electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood in view of the followingnon-limiting figures, in which:

FIG. 1 illustrates the principle of operation of an immunosensor inaccordance with some aspects of the invention;

FIG. 2 shows a side view of the fabrication of an immunosensor inaccordance with some aspects of the invention;

FIG. 3 shows an isometric view of a disposable sensing device and readerdevice in accordance with some aspects of the invention;

FIG. 4 shows an isometric top view of an immunosensor cartridge cover inaccordance with some aspects of the invention;

FIG. 5 shows an isometric bottom view of an immunosensor cartridge coverin accordance with some aspects of the invention;

FIG. 6 shows a top view of a tape gasket in accordance with some aspectsof the invention;

FIG. 7 shows an isometric top view of an immunosensor cartridge base inaccordance with some aspects of the invention;

FIG. 8 shows a schematic view of the layout of an immunosensor cartridgein accordance with some aspects of the invention;

FIGS. 9A-9E show top, bottom, side, and perspective views of animmunosensor cartridge in a closed position in accordance with someaspects of the invention;

FIG. 10 shows chronoamperometric data of cTnI in whole blood and urineon i-STAT® in accordance with some aspects of the invention;

FIG. 11 shows chronoamperometric data of urine and whole bloodbackground comparisons on i-STAT® showing analyte and reference currentsfor zero analyte cTnI samples in accordance with some aspects of theinvention;

FIG. 12 shows chronoamperometric data for NGAL in urine with and withoutexogenous albumin in sample processed on i-STAT® in accordance with someaspects of the invention;

FIG. 13 shows a response curve for an NGAL sandwich ELISA in accordancewith some aspects of the invention;

FIG. 14 shows a response curve for a competitive NGAL ELISA onmicrotitre plate in accordance with some aspects of the invention; and

FIG. 15 shows a response curve for a competitive NGAL ELISA with spikedrhNGAL in conditioned urine in accordance with some aspects of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention relates to analytical testing devices and methodsfor performing electrochemical immunoassays. More specifically, theinvention relates to analytical testing devices and methods forperforming electrochemical immunoassays on urine samples, and inparticular using point of care immunoassays for testing for analytes inurine samples. Some embodiments of the present invention may beimplemented by treating or amending a substantially undiluted or dilutedurine sample with a reagent comprising urease and/or other enzymes, awater soluble protein, a buffer, scavengers, or combinations thereof toadjust a pH of the amended urine sample to within a preselected rangefor an immunoassay binding reaction, and performing a quantitativeimmunoassay on the amended urine sample. The immunoassay may be aone-step immunoassay, a low wash immunoassay, or a homogenousimmunoassay.

Some aspects of the present invention are configured for rapid in situdeterminations of analytes using a cartridge, preferably a disposablecartridge, having an array of sensors (e.g., a pair of immunosensorscomprising a primary sensor and optionally a reference sensor) and ameans for sequentially or substantially sequentially presenting a sample(e.g., an amended urine sample) to the sensor array. The cartridges maybe configured to be preferably operated with a reading device, such asthe reading devices disclosed in U.S. Pat. No. 5,096,669, U.S. Pat. Nos.5,821,399, and 7,419,821, which are incorporated herein by reference intheir entireties. Details of the cartridge and reader device utilized inaccordance with these aspects of the present invention are describedbelow in further detail and may be best understood with reference to thecommercially available i-STAT® system for performing many differentassays including immunoassays.

One embodiment, therefore, of the present invention is directed to adevice configured to perform an immunoassay for a target analyte in aurine sample. The device may include a first region (e.g., a firstconduit or a first portion of a conduit) that comprises reagents, whichare configured to amend the urine sample. The reagents may comprise awater soluble protein and a buffer. The device may further include asecond region (e.g., a second conduit or a second portion of a conduit)comprising at least one immunosensor or electrode configured todetermine a concentration of the target analyte in the amended urinesample. The first region may be configured to provide a predetermineddissolved concentration of the water soluble protein to the urine sampleto form an amended urine sample. The predetermined dissolvedconcentration of the water soluble protein may be in a range of about0.02

to 225 mg/mL, e.g., from 0.02 to 200 or from 1 to 100.

In another aspect, the reagents may comprise urease and a buffer. Thefirst region may be configured to provide a predetermined dissolvedenzymatic activity within the amended urine sample. The predetermineddissolved urease enzymatic activity may be in a range of about 10 to10,0000 IU/mL, e.g., from 50 to 5000 or from 100 to 2000.

In another embodiment, the present invention is directed to a device forperforming a quantitative immunoassay for a target analyte in a urinesample. As used herein, the term “urine sample” refers to either adiluted or undiluted urine sample. The device may comprise a housingwith a first region for receiving a urine sample and amending the urinesample with a dissolvable reagent. The dissolvable reagent, preferablyurease combined with albumin and a buffer, may be coated onto a wall ofthe first region or added beforehand to the urine sample. The devicealso may have a second region connected to the first region, wherein thesecond region is configured to receive the amended urine sample and forman immobilized complex between the target analyte, an immobilized firstantibody, and an enzyme-labeled second antibody or an enzyme-labeledtarget analyte molecule.

In operation, therefore, one goal of the present invention is to providea device such as an immunosensor cartridge that is preferably operatedas follows. An unmetered amount of a biological sample (e.g., a urinesample) is placed into a sample chamber of the cartridge, and thecartridge is placed into a reading device. A metered portion of thesample is amended with a reagent comprising urease and/or other enzymes,a water soluble protein, a buffer, scavengers, or a combination thereof,and is then contacted with the at least one immunosensor or electrode.An electrical response of the at least one immunosensor or electrode isrecorded and analyzed for the presence, or amount of, an analyte ofinterest in the amended sample.

Amending Reagent

The general concept for treating or amending the urine sample in thepresent invention is based on the use of an amending reagent comprisingurease and/or another enzyme, a water soluble protein, a buffer,scavengers, or combinations thereof that are provided as a urine-solublesolid matrix, e.g., in a dried printed spot containing variousexcipients, such as sugars, to stabilize the dried reagent within aconduit of a device (e.g., a cartridge). For example, the reagent may bedisposed in a solubilizing agent, e.g., a sugar matrix, within a conduitin the device downstream of where the urine sample is introduced intothe device, but upstream of the at least one immunosensor or electrodesuch that when the urine sample contacts the solubilizing agent, thereagent is solubilized into the urine sample prior to performance of theimmunoassay at the at least one immunosensor or electrode.

In some embodiments, the enzyme(s) (e.g., urease) may be added to thereagent to counteract the potential damaging influence of the urinesample on the performance of the immunoassay. Urea is a common componentof urine and is known to be a strong protein denaturant that maypotentially cause the inactivation of enzymes by unfolding the proteinconstituting the enzyme into a non-functioning structure. Immunoassaystypically use an enzyme (e.g., alkaline phosphatase (ALP), horseradishperoxidase (HRP), and/or glucose oxidase (GOX)) conjugated to a highlyspecific binding species, such as, an antibody or aptamers for detectionof the desired analyte. The enzymes in these conjugates may besusceptible to the denaturing effects of urea present in urine sample.Accordingly, some aspects of the present invention include addingenzymes (e.g., urease) to the urine sample such that the ureaconcentration in the urine sample may be set and/or maintained below apreselected threshold value, which reduces the potential forinterference with detection enzyme (e.g., ALP) activity.

For example, single step immunoassays (e.g., ELISA) that may be used inthe present invention generally have both the capture antibody and thedetection antibody conjugate present in the same sample. Therefore, whenthe urine sample contains a denaturing agent, such as urea, the antibodyconjugate can lose enzyme activity, potentially providing a falsenegative or an analytical result that is less than the accurate value.On the other hand, two-step immunoassays function by a first step wherethe antigen present in a sample first binds to a capture antibody. Thesample and unbound antigen are then washed away from the captureantibody. For a urine sample containing high concentrations of urea, theurea is also washed away from the assay. A detection conjugate (whichmay comprise an enzyme) is then added to the assay, which will not beexposed to urea present in the urine sample. Thus, two-step immunoassaysare impacted less by the presence of urea in the urine, as it is washedaway in the process.

Moreover, thin layer chromatography assays (TLC) use a solvent thatdilutes the original sample, which may reduce the concentration of theurea present in a urine sample at the capture site of the TLC assay,along with the ability of the adsorbing material to bind the urea. Thus,TLC assays may be impacted less by the presence of urea in the urine, asthe urea is diluted in the process. Consequently, a one-stepimmunoassay, a low wash immunoassay, or a homogenous immunoassay mayadvantageously benefit from the removal of urea in a urine sample. Inaccordance with some aspects of the invention, immunoassay performanceis improved be removing or reducing the urea concentration.

More specifically, in one aspect of the invention, enzymes may beprovided to remove or reduce the urea concentration in the urine samplebelow a preselected threshold value such that the denaturing of proteinsused in the immunoassay may be avoided. One degradative enzyme of ureais urease [EC 3.5.1.5], which catalyzes the hydrolysis of urea withwater into carbon dioxide and ammonia. Urease functions best whenmercaptans like dithiothreitol (DTT) are present in the solution(Perlzweig, 1932, Science, vol 76:435-6). Therefore, mercaptans may alsobe a component of the reagent. In solution, urease, mercaptans, and ALPare compatible. However, mercaptans can poison a reference electrode(e.g., a Ag/AgCl reference electrode) in an electrochemicalimmunosensor. Therefore, in some embodiments, urease may be providedwith the lowest possible mercaptan concentrations possible as should beunderstood by those of ordinary skill in the art such that themercaptans do not interfere with the reference sensor. This may beaccomplished by dialyzing or column buffer exchanging the enzymepreparation.

It can be generalized that 1 IU of urease converts about 1 μMol of ureaper minute. A 3 μL sample of urine may contain about 0.4 M urea, whichwould contain approximately 12 μMol of urea. Thus, an excess of ureasemay be required to convert urea efficiently in less than a minute (e.g.,in test systems such as the present invention in which time is apredominant factor). Accordingly, in some embodiments, about 45-55 IU ofurease (preferably about 50 IU of urease) may be added to the reagent toprovide a dissolved urease enzymatic working range of about 10-10,000IU/mL.

In some embodiments, the urease may be provided to reduce the ureaconcentration of the urine sample below a preselected urea threshold,e.g., a preselected threshold of 10 mM. More preferably, the urease maybe provided to remove or reduce the urea concentration of the urinesample below the preselected urea threshold of 0.1 mM. Urease catalyzesthe following reaction:(NH₂)₂CO+H₂O

CO₂+2NH₃

In addition to enzymes that remove or reduce the urea concentration(e.g., urease), other enzymes may also be added to the reagent. Forexample, Metz et al., 1970, Clinica Chimica Acta, speculates that anincrease in ammonia in a sample when urea is present in an ALP assay maybe inhibitory for enzyme activity. Therefore, to overcome this potentialproblem, another enzyme(s) may be added in some embodiments of thepresent invention to remove the ammonia, e.g., as it is formed via theurease equilibrium reaction. One potential enzyme for this purpose isglutamic dehydrogenase [EC 1.4.1.2]. Other enzymes that consume ammoniaand generate less toxic substrates could also be used for this purpose(e.g., glutamine synthetase [EC 6.3.1.2] or any other urea cycle enzymeconfigured to consume ammonia). The reaction for glutamic dehydrogenaseis as follows:2-oxoglutarate+NH₃+NADH+H⁺

L-glutamate+H₂O+NAD.To assist glutamic dehydrogenase in converting NH₃ to L-glutamate,2-oxoglutarate and NADH may also be added to the reagent such that theyare provided in the reaction in excess to assist glutamic dehydrogenasein converting NH₃ to L-glutamate.

Furthermore, it should be noted that the use of glutamic dehydrogenase,glutamine synthetase, or other enzymes found in the urea cycle mayincrease a concentration of phosphate within the sample, which mayfurther inhibit enzyme (e.g., ALP) activity. Thus, it may beadvantageous to add an enzyme to the reagent that could sequester and/orreduce the concentration of excess phosphate in the sample. An exampleof such an enzyme would be adenylate kinase, which is used to regenerateadenosine triphosphate (ATP) from the adenosine diphosphate (ADP) andphosphate created by the action of enzymes such as glutamine synthetase.Accordingly, in some embodiments, the reagent may further comprise asequestering enzyme configured to sequester and reduce the concentrationof phosphate below a preselected phosphate threshold. Preferably, thepreselected phosphate threshold may be from 0 to 1 mM, or about 1/100the number of enzyme (e.g., ALP) molecules.

Another approach to improve immunoassay performance (e.g., the one-stepimmunoassay) may be to have an enzyme conjugate that is resistant todenaturing from the presence of urea in the urine sample. For example,neutrophil alkaline phosphatase (NALP) is resistant to urea (Cuckle etal., 1990, BMJ, 301:1024; Denier et al., 2002, BMC Biochemistry, 3(2):1)and may be used for the enzyme conjugate in place of the traditionalALP. Therefore, the use of a urea-resistant enzyme (e.g., NALP) in theimmunoassay system of the present invention may alleviate the inhibitionof the signal due to the denaturing effect of urea on the enzymeconjugate.

In some embodiments, a water soluble protein may be provided tosubstantially block non-specific binding of immunoglobulins at thesensor array and throughout conduits of the device. Additionally, thewater soluble protein may be provided to increase the viscosity of theurine sample and advantageously improve fluidic control within thedevice (e.g., improved control of movement of the urine sample from anentry port to a sensor array). In accordance with these aspects of theinvention, the water soluble protein may comprise forms of albumin andderivates thereof, forms of casein and derivatives thereof, or forms ofwhey proteins and derivatives thereof. In the instance of albumin, formssuch as, recombinant or non-recombinant human serum albumin, porcineserum albumin, castor bean albumin, salmon serum albumin, bovine serumalbumin, or a mixture thereof may be applicable to the aspects of thepresent invention. Albumin is a water soluble protein coagulated byheat, found in egg white, blood plasma and milk. Plants may also form asource of water soluble albumin proteins. Youle and Huang, (1978, PlantPhysiol., 61: 13) also indicate that there are high concentrations ofalbumins in many seeds. In particular, Youle and Huang found that aspecific albumin, 2S, constitutes 40% of the total castor seed protein.Serum Albumin is the predominant plasma protein in Atlantic Salmon(Salmo salar) (Xu & Ding, 2005, Biochemistry and Biotechnology, 35:283).The different forms of albumin can function as a chelator and bind someinterferents. Further, it is possible that the albumin can coat theimmunosensor to protect it from interferents.

In some embodiments, a buffer may be provided to adjust a pH of theurine sample to within a preselected range for the immunoassay bindingreaction. Specifically, it is known that the pH of urine samples mayvary widely. However, the urine sample is generally weakly buffered.Therefore, a buffer material may be added to the reagent to re-bufferthe urine into a desirable preselected range for the immunoassay bindingreaction. Preferably, a buffer may be selected that yields a pH in apreselected range of about pH 6 to pH 10.5. More preferably, a buffermay be selected that yields a pH in a preselected range of about pH 8.5to pH 9. In some aspects of the present invention, the buffer selectedmay be glycine, 3-(N-morpholino)propanesulfonic acid (MOPS),tris(hydroxymethyl)aminomethane (Tris), tricine, acetate, borate,2-(N-morpholino)ethanesulfonic acid (MES),2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES), or combinations thereof.

In some embodiments, scavengers may be provided to reduce non-specificcurrent generation derived from electroactive species. Morespecifically, the scavengers may comprise vitamin B scavengers to reducenon-specific current generation derived from electroactive vitamin Bspecies.

Amperometric Electrochemical System for the Detection of a TargetAnalyte

FIG. 1 illustrates the principle of an amperometric electrochemicalsystem according to specific embodiments of the present invention fordetermination of a NGAL 5, a marker for AKI. However, it should beunderstood that while specific embodiments are described for an NGALassay, the sensor structure and microparticle reagents described hereinmay also be useful for detecting human chorionic gonadotrophin, troponinI, troponin T, Chlamydia, Legionella, acetaminophen, amphetamines,methamphetamines, barbiturates, benzodiazepines, cocaine, methadone,opiates, phencyclidine, marijuana, tricyclic antidepressants, amongother analytes.

In a capture step, a sample, e.g., urine, may be introduced into aconduit or sample holding chamber of a cartridge of the presentinvention (e.g., a cartridge as disclosed in U.S. Pat. No. 7,723,099,which is incorporated herein by reference in its entirety) with afluidics format suitable for an immunoassay. In some embodiments, theurine sample is substantially undiluted. However, in additional oralternative embodiments, the urine sample may be diluted. For example, acomponent of the cartridge (as discussed in further detail herein) maybe configured to dilute the urine sample with a diluent. In some aspectsof the invention, the diluted urine sample may comprise from 5 to 95,from 10 to 90, or about 99% diluent.

In use, the enzymes may be provided in one or more layers, preferablydisposed within one or more conduits of the immunoassay device, e.g.,cartridge. For example, a first layer may be formed on a portion of awall of the conduit or the sample holding chamber of the cartridge. Thefirst layer may comprise the amending reagent (a discussed hereincomprising urease and/or one or more other enzymes, a water solubleprotein, a buffer, scavengers, or a combination thereof), which isconfigured to amend the urine sample. In addition, a second layer may beprovided and formed on a portion (a same or different portion as theportion having the reagent) of a wall of a conduit (a same or differentconduit as the conduit having the reagent) comprising a conjugatemolecule 10 labeled with an enzyme (e.g., ALP covalently attached to apolyclonal anti-NGAL (e.g., a signal antibody)), which is configured tobind to a target analyte (e.g., NGAL) within the urine sample. Forexample, the urine sample may be mixed with the amending reagent and theconjugate reagent, which are bound to a surface of the conduit or thesample holding chamber of the cartridge.

The conjugate molecule 10 may specifically bind to the NGAL 5, in theurine sample, producing a complex or conjugate comprising the NGAL 5bound to an ALP signal antibody 10. In a capture step, the complexcomprising the NGAL 5 bound to the ALP may bind to a capture NGALantibody 15 (e.g., an immobilized antibody) attached on, or close to, atleast one amperometric working electrode 20. Accordingly, the at leastone amperometric working electrode 20 may be coated with a biolayercomprising the covalently attached capture NGAL antibody 15, to whichthe complex comprising the NGAL 5 bound to the ALP conjugate molecule10. The ALP is thus immobilized on or in close proximity to the at leastone amperometric working electrode 20.

A capture region on the at least one amperometric working electrode 20may be defined by a hydrophobic ring of polyimide or anotherphotolithographically produced layer. A microdroplet or severalmicrodroplets (approximately 5-40 nanoliters in size) containingantibodies in some form, for example bound to latex microparticles, maybe dispensed on the surface of each sensor. The photodefined ringcontains this aqueous droplet allowing the antibody coated region to belocalized to a precision of a few microns. The capture region may bemade from about 0.03-2 mm² in size. The upper end of this size (e.g., 2mm²) may be limited by a size of a sensor conduit comprising the sensorsin present embodiments, and is not a limitation of the invention.

In addition to specific binding, the complex comprising the NGAL 5 boundto the ALP signal antibody 10 may also bind non-specifically to theelectrode. Non-specific binding may introduce a background signal fromthe electrode that is undesirable, and preferably should be minimized.Accordingly, in a rinsing step, a rinsing protocol (e.g., a low volumewash or limited wash) that utilizes a segmented fluid to rinse thesensors may provide an efficient means to minimize the backgroundsignal. In some embodiments, the limited wash may be less than fiftytimes a volume of the amended urine sample and/or fewer than threeindependent cycles of clean wash buffer (e.g., three independent washingsteps with fresh wash buffer) as should be understood by those ofordinary skill in the art of immunoassay procedures. In an analysis stepsubsequent to the rinsing step, a substrate 30 that is hydrolyzed by,for example, ALP to produce a detectable electroactive product 35 may beintroduced to the electrode. In specific embodiments, the substrate 30may be comprised of p-aminophenylphosphate or other suitable materialsuch as a phosphorylated ferrocene.

Thereafter, the ALP attached to the complex reacts with the substrate 30to form the detectable product 35 that may be indicative to aconcentration of the captured NGAL 5 within the urine sample. Thedetectable product 35 causes an electrical potential to be generatedacross the at least one amperometric working electrode 20 that in turngenerates a signal relative to the electrical potential caused by thedetectable product 35. The detectable product 35 generated from thereaction of the ALP with the substrate 30 at the at least oneamperometric working electrode 20 may be essentially proportional to anamount of NGAL 5 initially present in the urine sample.

In some embodiments, the substrate 30 may comprise a p-aminophenolspecies, and may be selected such that a voltammetric half-wavepotential (E_(1/2)) of the substrate 30 and the detectable electroactiveproduct 35 differ substantially. Preferably, the E_(1/2) of thesubstrate 30 is substantially higher (more positive) than that of theproduct 35. For example, when the E_(1/2) of the substrate 30 issubstantially higher (more positive) than that of the product 35, theproduct 35 can be selectively electrochemically measured in the presenceof the substrate 30.

The detection of ALP activity in the above example relies on ameasurement of the p-aminophenol oxidation current. This is achieved ata potential of about +20 to +60 mV, or from about +25 to +35 mV versusan optional Ag/AgCl ground or reference electrode. The exact form ofdetection used depends on the sensor configuration. In one embodiment ofthe sensor, an array of gold microelectrodes may be located directlybeneath the antibody capture region (e.g., the biolayer). When theanalysis fluid is pulled over the array of gold microelectrodes, enzymelocated on the capture site converts the p-aminophenylphosphate top-aminophenol in an enzyme-limited reaction. The concentration of thep-aminophenylphosphate may be selected to be in excess, e.g., 10 timesthe Km value. The analysis solution is 0.1 M in diethanolamine, 1.0 MNaCl, buffered to a pH of 9.8. Additionally, the analysis solution maycomprise 0.5 mM MgCl, which is a cofactor for the enzyme. Alternatively,a carbonate buffer has the desired properties and may be included in theanalysis fluid.

Amperometric Working Electrode Fabrication

Preferred embodiments of a microfabricated sensor array comprising atleast one amperometric working electrode is shown in FIG. 2. In thepreferred embodiments, the microfabricated sensor array may comprise apair of immunosensors or electrodes comprising a primary sensor orelectrode and optionally a reference sensor or electrode. For example,the immunosensors or electrodes may be fabricated as adjacentstructures, respectively, on a silicon chip.

In the preferred embodiments, the electrodes may be formed with goldsurfaces coated with a photodefined layer of polyimide. For example,wafer-level microfabrication of a preferred embodiment of the sensorarray may be achieved as follows. A planar non-conducting substrate 100may be used as a base for the sensor array. A conducting layer 105 maybe deposited on the substrate 100 by conventional means ormicrofabrication known to those of skill in the art to form at least oneelectrode. The conducting layer 105 may comprise a noble metal such asgold or platinum, although other unreactive metals such as iridium mayalso be used, as many non-metallic electrodes of graphite, conductivepolymer, or other materials may also be used.

For example, a base electrode may comprise a square array of 5-10 μmgold disks, e.g., 7 μm gold disks, on 15 μm centers. The array may covera region, e.g., a circular region, approximately 300 to 900 μm indiameter, optionally 600 μm in diameter, and may be formed byphoto-patterning a thin layer of the polyimide of thickness 0.35 μm overa substrate made from a series of layers comprising Si, SiO₂, TiW,and/or Au, or combinations thereof. The array of microelectrodes affordshigh collection efficiency of electroactive species with a reducedcontribution from any electrochemical background current associated withthe capacitance of the exposed metal. In particular, regularly spacedopenings in the insulating polyimide layer define a grid of small goldelectrodes at which the p-aminophenol may be oxidized in a 2 electronper molecule reaction.

Microfabrication techniques (e.g. photolithography and plasmadeposition) may be utilized for construction of the multilayered sensorstructures in confined spaces. For example, methods for microfabricationof the electrochemical immunosensors on silicon substrates are disclosedin U.S. Pat. No. 5,200,051, which is hereby incorporated by reference inits entirety. These include dispensing methods, methods for attachingbiological reagent, e.g., antibodies, to surfaces including photoformedlayers and microparticle latexes, and methods for performingelectrochemical assays.

The microfabricated sensor array may also comprise an electricalconnection 110 and a biolayer 115 (as discussed above with respect toFIG. 1), which are deposited onto at least a portion of the conductinglayer 105 and/or the non-conducting substrate 100. In the presentinvention, the biolayer 115 may include a porous layer comprising asurface with a sufficient amount of a molecule 120 (e.g., theimmobilized antibody and/or the microparticle reagent) that may eitherbind to an analyte of interest, or respond to the presence of such ananalyte by producing a change that is capable of measurement.

Optionally, a permselective screening layer may be interposed betweenthe conducting layer 105 and the biolayer 115 to screen electrochemicalinterferents as described in U.S. Pat. No. 5,200,051, which is herebyincorporated by reference in its entirety. In particular, the electrodesdescribed herein may be manufactured to optimize a signal-to-noiseratio, or amperometric background signal. For example, an interveningpolyvinyl alcohol (PVA) layer of about 0.5-5.0 μm thickness (preferably0.6-1.0 μm) may be placed between the electrodes and the biolayer orantibody reagent layer significantly attenuating the backgroundcomponent, as described in U.S. Pat. No. 7,723,099, which is herebyincorporated by reference in its entirety. An advantage of PVA as thebackground-reducing layer is that noise is reduced without appreciablyaffecting the Faradaic component of the signal. While the PVA layerreduces the diffusion coefficient of small molecules by about 50% it hasbeen found that it does not change the current at the coated electrodes,for two reasons. First, with PVA layers of about 1 micron thickness, thedetected electroactive species is present in a diffusion layer of atleast ten times that thickness, so there is little decrease in transportdue to the PVA layer. Second, a steady-state current is measured in theimmunosensor, which is effectively independent of the transport rate andelectrode kinetics, but is a function of the enzymatic rate ofproduction of the detectable species, such as p-aminophenol generatedfrom p-aminophenylphosphate by the enzyme ALP (attached to the signalantibody).

The porous PVA layer may be prepared by spin-coating an aqueous mixtureof PVA plus a stilbizonium photoactive, cross-linking agent over themicroelectrodes on the wafer. The spin-coating mixture optionallyincludes bovine serum albumin (BSA). The spin-coating mixture may thenbe photo-patterned to cover only a region above and around the sensorarrays, and preferably has a thickness of about 0.6 μm.

In specific embodiments, the biolayer 115 may be formed from latex beadsof specific diameter in the range of about 0.01 to 5.0 μm. The beads maybe modified by covalent attachment of any suitable molecule consistentwith the above definition of the biolayer (as discussed in furtherdetail below). Many methods of attachment exist in the art, includingproviding amine reactive N-hydroxysuccinimide ester groups for thefacile coupling of lysine or N-terminal amine groups of proteins. Inspecific embodiments, the molecule is an antibody selected to bind oneor more of NGAL, human chorionic gonadotrophin, troponin I, troponin T,Chlamydia, Legionella, acetaminophen, amphetamines, methamphetamines,barbiturates, benzodiazepines, cocaine, methadone, opiates,phencyclidine, marijuana, tricyclic antidepressants, or modifiedfragments thereof, more preferably NGAL. Such modified fragments aregenerated by oxidation, reduction, deletion, addition or modification ofat least one amino acid, including chemical modification with a naturalmoiety or with a synthetic moiety. Preferably, the molecule binds to theanalyte specifically and has an affinity constant for binding analyteligand of about 1×10⁻⁷ to 1×10⁻¹⁵.

In one embodiment, the biolayer 115 comprising microparticle beadshaving surfaces that are covalently modified by a suitable molecule, maybe affixed to the sensors by the following method. A microdispensingneedle may be used to deposit onto a surface of the electrode or aphoto-patterned PVA permselective layer covering the electrode a smalldroplet of the microparticle reagents. Specifically, in order to bindthe microparticle reagents to the electrode, a droplet of about 0.4 nLcomprising about 1% solids (i.e., the microparticles) in 0.08% Tween 20may be microdispensed (e.g., using the method and apparatus of U.S. Pat.No. 5,554,339, which is incorporated herein by reference in itsentirety) onto a surface of the electrode or a photo-patterned PVApermselective layer covering the electrode. The droplet may then beallowed to dry. The adherence of the dried microparticles particles tothe porous layer substantially prevents dissolution of themicroparticles into the sample (e.g., the urine sample) or the washingfluid. However, in some embodiments additional coupling chemistry may beused to ensure bead immobilization on the porous layer and/or theimmunosensors. Such techniques are well known in the art.

Microparticle Reagent Fabrication

In some embodiments, microparticles (e.g., carboxylate-modified latexmicroparticles supplied by Bangs Laboratories Inc. or SeradynMicroparticles Inc.) coated with antibodies (e.g., anti-NGAL andanti-HSA may be prepared for use in detecting target analytes such asNGAL in accordance with some aspects of the present invention. Forexample, the microparticles may first be buffer exchanged bycentrifugation, and then the antibodies may be added to themicroparticles (e.g., the antibodies may be allowed to passively adsorbonto the microparticles). In active groups (e.g., carboxyl groups) onthe microparticles may then be activated to form amide bonds to theantibodies (e.g., anti-NGAL and anti-HSA). Microparticle aggregates maythen be removed by centrifugation and the finished microparticles may bestored frozen for future use with the systems and devices of the presentinvention.

More specifically, NGAL capture/analyte beads may be prepared asfollows: 10 mg of 0.2 carboxylated microparticles (10% weight/volume)may be buffer exchanged into 25 mM 2-(N-morpholino)ethanesulfonic acid(MES, pH 6.2). The microparticles may then be reacted with 0.15 mganti-HSA mAb for 20 minutes at 4° C. with rotation. Subsequently, 0.8 mgof NGAL mAb may be added to the microparticle/anti-HSA mixture androtated at 4° C. for an additional 20 minutes, then centrifuged toremove the supernatant. After resuspension of the pellet in 25 mM MESbuffer (to achieve 2.5% wt microparticles), 6 mM carbodiimide (EDAC) maybe added to the sample and reacted for 2 hours at 4° C. This may then befollowed by centrifuging the sample and washing the pellet with ⅕physiological phosphate buffer twice. A formulated sample with 6.4%solids in ⅕ physiological phosphate buffer may then be further dilutedwith a protein stabilization solution to 3.2% solids. The formulatedsample may then be rotated at 4° C. for 20 minutes, aliquoted, andstored at −80° C. for future use.

NGAL reference bead preparation may be as follows: the process may bethe same as NGAL capture/analyte beads process except using only 0.8 mganti-HSA antibody in the reaction. The use of reference beads inimmunosensor manufacture and operation is described in U.S. Pat. No.7,732,099, which is incorporated herein by reference in its entirety, inwhich an immuno-reference sensor is used to subtract a signal arisingfrom non-specific binding of the signal antibody to the immunosensor.

Signal Antibody Conjugate Fabrication

In some embodiments, conjugates comprising an antibody (e.g., anti-NGAL)labeled with an enzyme may be prepared for use in detecting targetanalytes such as NGAL in accordance with some aspects of the presentinvention. Specifically, conjugate synthesis may comprise the following:NGAL conjugate preparation may use 1.4 mg pepsin digested whole antibodyto make F(ab)2′ in 0.1 M citrate buffer (pH 3.5) at 37° C. for 45minutes (0.004 mg pepsin to 1 mg whole Ab). The pepsin digest may bestopped by the addition of Trizma base until the pH of the solution isadjusted to 7.2. The sample may then be cooled at 4° C. for 1 hour.Purification of the F(ab)2′ fraction may be performed by using a HiPrep16/60 Sephacryl S-300 High Resolution size exclusion column.Monoethanolamine hydrochloride (MEA) in ⅕ physiological phosphate buffermay be used to reduce F(ab)2′ to Fab-SH (final concentration is 6 mg/mLMEA) for 1 hour at 37° C. The Fab-SH may then be reacted with activatedsingle molecule alkaline phosphatase (ALP) in a 3:1 molar ratio at 4° C.overnight. ALP may previously be activated in the presence of LC-SMCC(Succinimidyl-4-[N-Maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate]for 40 minutes at room temperature) at a 10 LC-SMCC:1 ALP molar ratio.The Fab-SH/ALP mixture may then be quenched with 50 mM Tris inconjugation buffer at 4° C. for 1 hour. A size exclusion column may thenbe used to purify the conjugate fraction and formulate it into a ⅕physiological phosphate-buffered protein stabilization solution. ThisNGAL conjugate may then be stored at 4° C. for future use.

Competitive ELISA ALP-NGAL Tracer Fabrication

In some embodiments, activation of alkaline and rhNGAL proteins may becarried out prior to final conjugation. To thiolate the alkalinephosphatase, 10 mg of AP in 0.56 mL buffer (5 mM Tris, 50% glycerol, 5mM MgCl₂, 1 mM ZnCl₂) may be exchanged into activation buffer (PBS+10 mMEDTA, pH 7.4) using a 10K MWCO Amicon spin tube. The protein may then besuspended in 1 mL final volume of PBS 10 mM EDTA, pH 7.4 and reactedwith 35 μL of 2-Iminothiolane (3.5 mg/mL DMSO stock). The solution maythen be nutated for 1 hour at room temperature. The reaction may then bewashed 3 times and exchanged into 1 mL coupling buffer (PBS pH 7.4)using a 10K MWCO Amicon filter.

Maleimide activation of rhNGAL may be carried out by addition of 16 μLsulfo-SMCC (1 μg/μL stock in DMSO) to 77 μg rhNGAL in PBS pH 7.4(reaction volume 316 μL total). The reaction may be nutated for 2 hoursat room temperature. The reaction may then be washed 3 times andexchanged into 200 μL coupling buffer (PBS pH 7.4) using a 10K MWCOAmicon filter.

Final conjugation of activated AP to activated rhNGAL may be carried outby adding 26 μL activated AP from above into a 200 μL activated rhNGALvial. The reaction may be nutated at room temperature for 1 hour andthen allowed to proceed to completion at 4° C. overnight. The rhNGAL-APconjugate (NGAL tracer) may then be washed 3 times with TBS, pH 8 usingan Amicon 100K MWCO filter tube which enabled removal of unreactedrhNGAL (˜25000 kDa). The NGAL tracer may then be suspended in 231 μL 1%BSA in TBS. Any dilutions of the tracer used for off-cartridgeimmunoassay may then be made in 1% BSA in TBS, while dilutions foron-cartridge assays may be made directly in donor urine.

System Comprising a Sensor Array Configured for Target Analyte Detection

Referring to FIG. 3, the system 300 of the present invention maycomprise a self-contained disposable sensing device or cartridge 301 anda reader device or instrument 302. A fluid sample (e.g., urine) to bemeasured is drawn into a sample entry orifice or port 303 in thecartridge 301, and the cartridge 301 may be inserted into the readerdevice 302 through a slotted opening 304. The reader device 302 maycomprise a processor configured to perform measurements of analyteconcentration within the fluid sample, as discussed herein in furtherdetail. Measurements and determinations performed by the reader may beoutput to a display 305 or other output device, such as a printer ordata management system 307 via a port on the reader 308 to a computerport 309. Transmission can be via Wifi, Bluetooth link, infrared and thelike. Note that where the sensors are based on electrochemicalprinciples of operation, the sensors 310 (e.g., a primary sensor andoptionally a reference sensor) in the cartridge 301 make electricalcontact with the instrument 302 via an electrical connector 311. Forexample, the connector may be of the design disclosed in jointly ownedU.S. Pat. No. 4,954,087, incorporated herein by reference in itsentirety. The instrument 302 may also include a method for automaticfluid flow compensation in the cartridge 301, as disclosed in jointlyowned U.S. Pat. No. 5,821,399, which also is incorporated herein byreference in its entirety.

In some aspects of the invention, the cartridge 301 may be provided witha barcode with factory set information including equations to be usedand required test coefficients. The reader device 302, into which thecartridge 301 is inserted to run the test, may thus be equipped with abarcode reader. A selection of equations may be embedded in software ofthe reader device 302. For example, the coefficients for the cartridge301 may differ, where different lots of cartridges 301 are manufactured,each lot having slightly different factory-determined characteristics.In any event, the coefficients for the cartridge 301, from whichevermanufacturing lot the cartridge 301 is drawn, are conveyed to the readerdevice 302 for use in one or more of the equations, for that particularcartridge test. For example, if a given digit of the cartridge barcodeis set to 1, the reader device 302 may set a predetermined coefficientto zero, whereas other digits may code for different coefficients orselect a kinetic model to be used, e.g., an immunoassay model formulatedby analogy to the well-known Michaelis-Menton enzyme kinetics.

In one embodiment, as shown in FIGS. 4-7, a cartridge 400 (e.g., adisposable assay cartridge) may comprise a cover 405 (as shown in FIGS.4 and 5), a base 410 (as shown in FIG. 7), and a thin-film adhesivegasket 415 (as shown in FIG. 6) that is disposed between the base 410and the cover 405. The cartridge 400 may be configured for insertioninto a reader device, and therefore the cartridge 400 may comprise aplurality of mechanical and electrical connections (not shown) for thispurpose. Advantageously, a feature of the cartridge 400 is that once asample is loaded within the cartridge 400, analysis of the sample may becompleted and the cartridge 400 may discarded without an operator orothers contacting the sample.

Referring to FIG. 4, the cover 405 may be made of a rigid material,preferably plastic, and capable of repetitive deformation at flexiblehinge regions 420, 425, and 430 without cracking. The cover 405 maycomprise a lid 435, attached to a main body of the cover 405 by theflexible hinge 425. In operation, after introduction of a sample into asample holding chamber 440 (as shown in FIG. 7) through a sample entryport 445, the lid 435 may be secured over an entrance to the sampleentry port 445, preventing sample leakage. The lid 435 may be held inplace by a hook 450.

The cartridge 400 optionally may also have a closure feature asdescribed in jointly owned U.S. Pat. No. 7,682,833, which is herebyincorporated by reference in its entirety, for sealing the sample entryport 445 in an air-tight manner. This closure device may be slidablewith respect to a body of the cartridge 400 and provides a shearingaction that displaces excess sample located in the region of the sampleentry port 445, reliably sealing a portion of the sample in the sampleholding chamber 440 between the sample entry port 445 and a capillarystop. Specifically, the cartridge 400 may be sealed by slidably moving asealing element over the surface of the cartridge in a manner thatdisplaces excess fluid sample away from the sample entry port 445, sealsa volume of the fluid sample within the internal fluid sample holdingchamber 440, and inhibits fluid sample from prematurely breaking throughthe internal capillary stop.

The cover 405 may further comprise two paddles 455 and 460 that aremoveable relative to the body of the cover 405, and which are attachedto the cover 405 by the flexible hinge regions 420 and 430. The paddle460 may be configured to be operated by a pumping means such that aforce is exerted upon an air bladder comprised of cavity 465 (as shownin FIG. 7) and the gasket 415. Operation of the paddle 460 displacesfluid within conduits of the cartridge 400.

The paddle 455 may be configured to be operated upon by a second pumpingmeans such that a force is exerted upon the gasket 415, which can deformbecause of slits 470 cut therein (as shown in FIG. 6). Deformation ofthe gasket 415 may transmit pressure onto a fluid-containing foil packfilled with a fluid, e.g., approximately 130 μL of analysis/washsolution or fluid, located in cavity 475 (as shown in FIG. 7), rupturingthe foil pack upon spike 480, and expelling fluid into conduit 485. Theconduit 485 may be connected via a short transecting conduit in the base410 to a conduit 490 (as shown in FIG. 5). The fluid fills a front ofthe conduit 485 first pushing fluid into a small opening in the gasket415 that acts as a capillary stop.

Additional action in the cartridge 400 generated by mechanisms withinthe reading device applied to the cartridge 400 may be used to injectone or more air segments into the fluid at controlled positions withinthe conduit 490. The air segments may be used to wash a sensor surfaceof the sensor array and the surrounding conduit 490 with a minimumamount of fluid (e.g., a limited wash cycle in which the volume of washmay be less than fifty times a volume of the amended urine sample and/orfewer than three independent cycles of clean wash buffer (e.g., threeindependent washing steps with fresh wash buffer) as should beunderstood by those of ordinary skill in the art of immunoassayprocedures. For example, the cover 405 may further comprise a holecovered by a thin pliable film 495. In operation, pressure exerted uponthe film 495 may expel one or more air segments into the conduit 490through a small hole 505 in the gasket 415 (as shown in FIGS. 5 and 6).

Referring to FIG. 6, a lower surface of the cover 405 further comprisesthe conduit 490 and another conduit 510. The conduit 490 includes aconstriction 520 that controls fluid flow by providing resistance to theflow of the fluid. Optional coatings 525 and 530, e.g., dry reagentcoatings, may provide hydrophobic surfaces on the conduit 510, whichtogether with gasket holes 535 and 540 control fluid flow betweenconduits 190 and 510. A recess 545 in the base may provide a pathway forair to enter and/or escape the conduit 440 through hole 550 in thegasket.

Referring to FIG. 6, the thin-film gasket 415 comprises various holesand slits to facilitate transfer of fluid and air between conduitswithin the base 405 and the cover 410, and to allow the gasket 415 todeform under pressure where necessary. Specifically, a hole 555 maypermit fluid to flow from the conduit 490 into a waste chamber 560, ahole 565 may comprise a capillary stop between conduits 440 and 510, ahole 570 may permit air to flow between a recess 575 (as shown in FIG.5) and a conduit 580 (as shown in FIG. 7), the hole 550 provides for airmovement between the recess 545 and the conduit 440, and the hole 505permits fluid to flow from a conduit 585 (as shown in FIG. 5) to thewaste chamber 560 via optional closeable valve 590 (as shown in FIG. 7).Holes 595 and 600 permit a plurality of electrodes (e.g., the primarysensor and optionally the reference sensor) that are housed withincutaways 605 and 610, respectively, to contact fluid within the conduit490. In a specific embodiment, cutaway 610 houses a ground electrode,and/or a counter-reference electrode, and cutaway 605 houses at leastone analyte sensor (e.g., the primary sensor), and optionally, areference sensor.

Referring to FIG. 7, the conduit 440 may be configured as a sampleholding chamber that connects the sample entry port 445 to the conduit510 in the assembled cartridge 400. The cutaway 605 may house at leastone analyte sensor (e.g., the pair of electrodes), or an analyteresponsive surface, together with an optional conductimetric sensor orsensors. The cutaway 610 may house a ground electrode if needed as areturn current path for an electrochemical sensor, and may also house anoptional conductimetric sensor. A cutaway 615 may provide a fluid pathbetween gasket holes 535 and 540 such that fluid may pass between theconduits 490 and 510. Recess 475 houses a fluid-containing package,e.g., a rupturable pouch, in the assembled cartridge 400 that may bepierced by the spike 480 because of pressure exerted upon paddle 455upon insertion of the cartridge 400 into the reading device. Fluid fromthe pierced package flows into the conduit 485. The air bladder may becomprised of the recess 465, which is sealed on its upper surface by thegasket 415. The air bladder may be one embodiment of a pump means, andmay be actuated by pressure applied to the paddle 460, which displacesair in the conduit 580 and thereby displaces the sample from the samplechamber 440 into the conduit 510.

In some embodiments, a metering means may optionally comprise the samplechamber 440 bounded by the capillary stop 565 and having along thechamber 440 length an air entry point (gasket hole 550) from thebladder. Air pressure exerted at the gasket hole 550 drives a meteredvolume of the sample past the capillary stop 565. Therefore, a meteredvolume of sample may be predetermined by a volume of the sample chamber440 between the air entry point 550 and the capillary stop 565. Anamount of the sample corresponding to this volume may be displaced intothe conduit 510 when the paddle 460 is displaced. This arrangement maytherefore provide a metering means for delivering a metered amount of anunmetered sample into the various downstream conduits of the cartridge400. The metering may be advantageous in some embodiments ifquantitation of the analyte is required. Thus, an operator may berelieved of accurately measuring the volume of the sample prior tomeasurement saving time, effort, and increasing the accuracy andreproducibility.

As shown in FIG. 8, a schematic diagram of the features of the cartridge700 and components therein is provided. Specifically, in preferredembodiments, the conduits and the sample chamber 705-735 may be coatedwith dry reagents to amend the sample or fluid as discussed herein. Thesample or fluid may be passed at least once over the dry reagent todissolve the dry reagent. Reagents that may be used to amend samples orfluid within the cartridge include urease and/or other enzymes, a watersoluble protein, a buffer, scavengers, or combinations thereof,antibody-enzyme conjugates, and/or blocking agents that prevent eitherspecific or non-specific binding reactions among assay compounds. Asurface coating that may not be soluble but helps prevent non-specificadsorption of assay components to the inner surfaces of the cartridge700 may also be provided.

For example, within a segment of the sample or fluid, an amendingsubstance may be preferentially dissolved and concentrated within apredetermined region of the segment. In one embodiment, this may beachieved through control of the position and movement of the segmentwithin the conduits and the sample chamber 705-735. Therefore, if only aportion of a segment, such as the leading edge, is reciprocated over theamended substance, then a high local concentration of the substance canbe achieved close to the leading edge. Alternatively, if a homogenousdistribution of the substance is desired, for example if a knownconcentration of an amending substance is required for a quantitativeanalysis, then further reciprocation of the sample or fluid may resultin mixing and an even distribution.

In preferred embodiments, a closeable valve 740 may be provided betweena first conduit and the waste chamber. In one embodiment, the valve 740may be comprised of a dried sponge material that is coated with animpermeable substance. In operation, contacting the sponge material withthe sample or a fluid may result in swelling of the sponge to fill thecavity (e.g., the valve 590 cavity as shown in FIG. 7), therebysubstantially blocking further flow of liquid into the waste chamber.Furthermore, the wetted valve 740 may also be configured to block theflow of air between the first conduit and the waste chamber, whichpermits a first pump means connected to the sample chamber to displacefluid within a second conduit, and to displace fluid from the secondconduit into the first conduit in the following manner.

After the sample is exposed to the sensor array (e.g., the primaryelectrode and optionally the reference electrode) for a controlled time,the sample may be moved into a post-analytical conduit where the samplemay be amended with another reagent. The sample may then be moved backto the sensor array and a second reaction period may begin. Alternately,the post-analysis conduit may serve simply to separate the samplesegment from the sensor array. Within the post-analysis conduit may be asingle closeable valve that connects an air vent of the sensor conduitto a diaphragm air pump. When the single closeable valve closes, thesample may be locked in the post analytical conduit and cannot be movedback to the sensor array. Such closable valves are described in U.S.Pat. No. 7,682,833, the entirety of which is incorporated herein byreference. In some embodiments, one or more air segments may be injectedinto the sample to facilitate washing using an active feature, e.g., apump, or a passive feature, e.g., a tape gasket, as described in U.S.Pat. No. 8,309,364, the entirety of which is incorporated herein byreference.

In a preferred embodiment of the present invention, the sample and afluid, e.g., a combined wash and enzyme substrate delivery fluid, maycontact the sensor array (e.g., the pair of electrodes and optionallythe reference electrode) at different times during an assay sequence.The sample and the fluid may also be independently amended with otherreagents or compounds present initially as dry coatings withinrespective conduits of a test device, e.g., the cartridge. Controlledmotion of the fluid by the above-described pumping means within thecartridge further permits more than one substance to be amended intoeach fluid whenever the sample or the fluid is moved to a new region ofthe conduit. In this manner, multiple amendments to each fluid may beaccommodated, extending the complexity of automated assays that can beperformed in the cartridge. Therefore, the utility of the presentinvention may be enhanced.

In an alternative embodiment, as shown in FIGS. 9A-9E, the cartridge 900may include a housing that comprises two complimentary halves of acartridge (e.g., the cover 901 and the base 902), which can be bondedtogether to abut and attach the two complimentary interior surfaces ofthe two halves in a closed position. In some embodiments, the cover 901and the base 902 are injection molded, for example, by machine asdisclosed in U.S. patent application Ser. No. 13/530,501, filed on Jun.22, 2012, which is incorporated herein by reference in its entirety.Preferably, the cover 901 is injection molded where a firstsubstantially rigid zone 920 is formed in a first injection molding stepand a substantially flexible zone 922 is formed in an additionalinjection molding step. Preferably, the base 902 is injection moldedwhere a second substantially rigid zone 924 is formed in a firstinjection molding step.

As shown in FIGS. 9A-9E, the substantially rigid zones 920 and 924 ofthe cover 901 and the base 902, respectively, are preferably each asingle contiguous zone; however, the molding process can provide aplurality of non-contiguous substantially rigid zones. The substantiallyflexible zone 922 is preferably a set of several non-contiguous zones.For example, the substantially flexible zone 922 around a displaceablemembrane 925 may be separate and distinct from the substantiallyflexible zone at a closeable sealing member 928. Alternatively, thesubstantially flexible zone may comprise a single contiguous zone.

In a preferred embodiment, the cartridge housing comprises a sensorrecess 930 in a portion of the substantially flexible zone. An advantageis that the sensors 935 (e.g., the primary sensor and optionally thereference sensor preferably each of a size of about 0.3×0.4 cm), whichare disposed in the sensor recess 930 preferably are made on a siliconwafer substrate, which is relatively brittle. Thus, providing asubstantially flexible sensor recess 930 results in a suitable supportthat can protect the sensor from cracking during assembly. Note thatother non-silicon based sensors may be used, e.g., those made on aplastic substrate; however, the preferred embodiment uses sensors of thetype described in U.S. Pat. Nos. 5,200,051; 5,514,253; and 6,030,827,the entireties of which are incorporated herein by reference. Inaddition to being substantially flexible, sensor recess 930 may be bestselected to form a liquid-tight and/or air-tight seal around the sensorperimeter, thereby ensuring that liquids do not leak out of the conduitthat covers the sensor in the fully assembled cartridge. In analternative embodiment, sensor recess 930 can be formed in a portion ofthe substantially rigid zone (as shown in FIG. 7) of either or both ofthe cover or the bottom of the housing. In this aspect, a liquid-tightand/or air-tight seal optionally may be formed by the double-sidedadhesive sheet 415 or gasket (as shown in FIG. 6).

With regard to overall dimensions, the preferred embodiment of themolded parts shown in FIGS. 9A-9E include the cover 901 with dimensionsof about 6.0 cm×3.0 cm×0.2 cm and the base 902 with dimensions of about5.0 cm×3.0 cm×0.2 cm to provide a cartridge 900 with dimensions of about6.0 cm×3.0 cm×0.4 cm. In terms of ranges, the cartridge 900 optionallyhas a length of from 1 to 50 cm, e.g., from 5 to 15 cm, a width of from0.5 to 15 cm, e.g., from 1 to 6 cm, and a thickness of from 0.1 to 2 cm,e.g., from 0.1 to 1 cm.

Processes for Target Analyte Detection in a Urine Sample

In preferred embodiments, the invention is a process for using acartridge to determine the presence and/or concentration of a targetanalyte in a urine sample. The process may include introducing anunmetered fluid urine sample into the sample chamber 440 of thecartridge 400 through the sample entry port 445 (as shown in FIGS. 4-7).Capillary stop 565 prevents passage of the urine sample into conduit 510at this stage, and conduit 440 is filled with the sample. Lid 435 isclosed to prevent leakage of the sample from the cartridge. Thecartridge may then be inserted into the reading device or apparatus 302,as shown in FIG. 3 and further disclosed in U.S. Pat. No. 5,821,399,which is incorporated herein by reference in its entirety. Insertion ofthe cartridge into the reading apparatus activates a mechanism, whichpunctures the fluid-containing package located at recess 475 when thepackage is pressed against spike 480. Fluid is thereby expelled into theconduits 485 and 490, arriving in sequence at the sensor region. Theconstriction 520 prevents further movement of fluid because residualhydrostatic pressure is dissipated by the flow of fluid via the conduit585 into the waste chamber 560.

In a second step, operation of a pump means applies pressure to theair-bladder comprised of cavity 465, forcing air through the conduit 580and into conduit 440 at a predetermined location. Capillary stop 565delimits a metered portion of the original sample. While the sample iswithin sample chamber 440, it is preferably amended with a compound orcompounds (e.g., urease and/or other enzymes, a water soluble protein, abuffer, scavengers, or a combination thereof, and/or antibodies to NGALlabeled with ALP) present initially as a dry coating or layer(s) on theinner surface of the chamber or conduits. The metered portion of thesample is then expelled through the capillary stop 565 by air pressureproduced within air bladder comprised of cavity 465. The sample passesinto the sensor conduit and into contact with the pair of electrodes andoptionally the reference electrode located within the cutaway 605.

To promote binding of the analyte, e.g., NGAL to the electrodes, thesample containing the analyte may optionally be passed repeatedly overthe electrodes in an oscillatory motion. Preferably, an oscillationfrequency of between about 0.2 and 2 Hz is used, most preferably 0.7 Hz.After a period, e.g., 10 minutes, for the analyte/enzyme-antibodyconjugate complex to bind to the electrodes, the sample may be ejectedby further pressure applied to the air bladder comprised of cavity 465,and the sample passes to waste chamber 560. A wash step (in someembodiments a limited wash step) next removes non-specifically boundenzyme-conjugate from the sensor chamber. Fluid in the conduit 490 maybe moved by a pump means, into contact with the sensors. The analysisfluid may be pulled slowly until a first air segment is detected at aconductivity sensor. Note that it may be an object of the invention thatthe rinsing is not sufficiently protracted or vigorous as to promotedissociation of specifically bound analyte or analyte/antibody-enzymeconjugate complex from the sensors.

Use of a cartridge with a closeable valve, preferably located betweenthe sensor chamber and the waste chamber, is herein illustrated by aspecific embodiment in which the concentration of NGAL is determinedwithin a urine sample, which is introduced into the sample chamber ofsaid cartridge. In the following time sequence, time zero (t=0)represents the time at which the cartridge is inserted into thecartridge reading device. Times are given in minutes. Between t=0 andt=1.5, the cartridge reading device makes electrical contact with theelectrodes/sensors through pads, and performs certain diagnostic tests.Insertion of the cartridge perforates the foil pouch introducing fluidinto a conduit as previously described. The diagnostic tests determinewhether fluid or sample is present in the conduits using theconductivity electrodes; determine whether electrical short circuits arepresent in the electrodes; and ensure that the sensor and groundelectrodes are thermally equilibrated to, preferably, 37° C. prior tothe analyte determination.

Various options exist for managing any temperature effect on animmunoassay of this type. For example, the assay can be run in a systemwhere the sample and other fluids and reagents are thermostated at agiven temperature, e.g., 37° C. Alternatively, the assay may be run atambient temperature, without any correction, or with correction to astandardized temperature based on measurement of the ambient value

Between t=1.5 and t=6.75, a metered portion of the urine sample,preferably between 4 and 200 μL, more preferably between 4 and 20 μL,and most preferably 7 μL, may be used to contact the electrodes/sensorsas described above. The edges defining the forward and trailing edges ofthe sample are reciprocally moved over the sensor region at a frequencythat is preferably between 0.2 to 2.0 Hz, and is most preferably 0.7 Hz.During this time, the amending reagent and enzyme-antibody conjugatedissolves within the sample, as previously described. The amount ofenzyme-antibody conjugate that is coated onto the conduit is selected toyield a concentration when dissolved that is preferably higher than thehighest anticipated NGAL concentration, and is most preferably six timeshigher than the highest anticipated NGAL concentration in the sample.

Between t=6.75 and t=10.0 the sample may be moved into the waste chambervia the closeable valve, preferably wetting the closeable valve andcausing it to swell and close. The seal created by the closing of thevalve permits the first pump means to be used to control motion of fluidfrom the sensor conduit to the post analysis conduit. After the valvecloses and any remaining sample is locked in the post analysis conduit,the analyzer plunger retracts from the flexible diaphragm of the pumpmeans creating a partial vacuum in the sensor conduit. This forces theanalysis fluid through the small hole in the tape gasket and into ashort transecting conduit in the base. The analysis fluid is then pulledfurther and the front edge of the analysis fluid is oscillated acrossthe surface of the sensor chip in order to shear the sample near thewalls of the conduit. A conductivity sensor on the sensor chip may beused to control this process. The efficiency of the process may bemonitored using the amperometric sensors through the removal of unboundenzyme-antibody conjugate which enhances the oxidation current measuredat the electrode when the enzyme substrate, p-aminophenyl phosphate isalso present. The amperometric electrodes may be polarized to 0.06 Vversus the silver chloride reference-ground electrode. In thisembodiment, the fluid may be composed of a 0.1 M carbonate ordiethanolamine buffer, at pH 9.8, with 1 mM MgCl2, 1.0 M NaCl, 10 mMp-aminophenylphosphate, and 10 μM (micromolar) NaI. The efficiency ofthe wash is optimally further enhanced by introduction into the fluid ofone or more segments that segment the fluid within the conduit aspreviously described. Following removal of wash fluid from the sensorchannel leaving a thin layer of fluid over the two sensors, measurementof each sensor response is recorded and the concentration of analytedetermined as described above.

EXAMPLES

For purposes of illustration and not limitation, the following examplesprovide information on the performing an immunoassay on a urine sampleand some aspects of the present invention including the amendment of theurine sample with urease and/or other enzymes, a water soluble protein,a buffer, scavengers, or combinations thereof.

Example 1 (i-STAT® Sandwich ELISA with cTnI in Urine)

Initial testing of urine as a sample matrix on the i-STAT platform wasperformed to compare cTnI spike results in urine and in whole blood. Asa proof of concept, preexisting cTnI i-STAT ELISA technology andmethodology was utilized prior to creating new NGAL methodologies.

cTnI i-STAT cartridges were warmed to room temperature from beingrefrigerated. Whole blood was drawn into a lithium heparin vacutainer,and midstream urine from the same donor was collected into a sterilevial (urine pH was ˜6.9). A manufacturer's working calibrator of cTnIwas used to spike whole blood or urine as appropriate (stock cTnI, 48.25ng/mL; dilutions into whole blood or urine with final concentrations of0.48, 1.86 and 9.65 ng/mL). It was determined that the donor's startinglevel of whole blood troponin was 0.0 ng/mL. Spiked samples were mixedon a nutator (Clay Adams, Parsippany, N.J.) at room temperature untiltime of use. Spiked whole blood and urine samples were loaded (20 μL) asreplicates onto temperature equilibrated cartridges and after the testcycle was completed, chronoamperometric data was analyzed to calculatetroponin levels. In addition, traces corresponding to motor motion,conductivity, reference and analyte current signals etc. were comparedbetween whole blood and urine-based samples. No fluidic movement errorcodes during the run cycle indicated that the i-STAT system was capableof moving urine as a sample type within the cartridge conduits.

FIG. 10 illustrates a typical comparison of amperometric data from wholeblood versus urine in this experiment. The signals generated from theurine were reproducibly almost half that of the whole blood samples.This data suggested an interference manifested by the presence of theurine sample. Without being bound by theory, possible mechanisms couldbe due to inadequate cTnI being recovered from the urine matrix duringthe ELISA steps or that the difference in conductivity between thesample types manifested an effect in signal or the urine containsinterferents of the assay.

In addition, background current from both the analyte and referenceelectrodes was higher in urine samples than in the whole blood samplesduring capture. FIG. 11 is chronoamperometric data of urine and wholeblood background comparisons on i-STAT showing analyte and referencecurrents for zero cTnI samples. Whole blood was represented byconsistently lower traces while urine was represented by elevatedsignals. This background current interference occurred consistentlythroughout the test cycle on the urine reference sensor indicating thatthe cause may have originated from the sample when the blocking stepshould have occurred (reference sensor in the i-STAT immuno cartridgesshould capture human serum albumin from the blood; in addition, the cTnIanalyte sensor beads also have anti-HSA capture antibody which shouldcapture HSA and reduce background). In general, the HSA in the bloodfunctions as an inherent blocking agent for the whole blood ELISA in thei-STAT system; serum albumin can also act as a buffer in solutions,especially where pH is at extremes (e.g., less than 3 and greater than10). Due to the orders of magnitude difference in the albumin/proteinconcentration in whole blood versus urine (avg. 45 g/L vs 0.02 g/L,respectively), improper blocking of cartridge sensors and plastic fromthe urine matrix components likely led to increased background signalswith urine.

Addition of casein, HSA or other albumin like BSA or recombinant humanserum albumin into the urine sample as a pretreatment may mitigate thistype of interference. Recombinant human serum albumin (rHSA) wasselected for use since it is specific for the capture antibody beingused in this example, and to reduce use of biohazardous material in thecartridge. Other interferents in urine (as compared to blood) can alsoplay a role in background signal on both or either of the reference andanalyte electrodes. For instance, certain B vitamins are known to beelectroactive and may contribute to amperometric signals during thei-STAT cartridge cycle. Vitamin B scavengers may be incorporated in aurine pretreatment step as well in order to mitigate this type ofelectroactive interferent.

Urine was successfully used as a sample type on the i-STAT sandwichELISA platform. Without being bound by theory, the background signal inurine seen on the reference and analyte sensors may be due to urineinterferents or insufficient protein from urine, which can help inimmunoassay systems.

Example 2 (i-STAT Sandwich Immunoassay with cTnI in rHSA-Amended Urine)

Commercially available cTnI i-STAT cartridges (Abbott Point of Care,Princeton, N.J.) were utilized in determining if addition of rHSA tourine samples would mitigate background current within the i-STATimmunoassay test cycle and increase analyte sensor current in urine.

A manufacturer's working calibrator of cTnI was used as described inExperiment 1 to spike urine as appropriate (stock cTnI, 48.25 ng/mL;dilutions into urine with final concentrations of 0.48, 1.86 and 9.65ng/mL). HSA solution was made up as a 225 mg/mL stock in urine; dilutioninto the test samples was to 45 mg/mL, corresponding to the average HSAconcentration found in whole blood). Spiked samples were mixed on anutator (Clay Adams, Parsippany, N.J.) at room temperature for 10minutes. 20 μL of spiked urine samples (+/−cTnI and +/−HSA) were loadedas replicates onto equilibrated cartridges and after the test cycle wascompleted, chronoamperometric data was analyzed to calculate troponinlevels. In addition, traces corresponding to motor motion, conductivity,reference and analyte current signals etc. were reviewed. No fluidicmovement error codes were logged during the run cycle, indicating thatthe i-STAT system was capable of moving urine +/−HSA as a sample typewithin the cartridge conduits. Background noise on reference and analytesensors during ELISA capture was aptly mitigated by addition of HSA to45 mg/mL. Adding HSA to urine also improved recovery of cTnI to almostthe same level as whole blood spiked with an identical amount oftroponin. Finally, adding HSA to urine spiked with low to medium levelsof cTnI (up to ˜2 ng/mL) mitigated background noise on the referencesensor during analysis, but had little effect with higher cTnI samples.

Example 3 (i-STAT Sandwich Immunoassay with NGAL in rHSA-Amended Urine)

Subsequent testing of urine as a sample type on the i-STAT platform wasperformed to compare rhNGAL (recombinant human) levels in urine+/−rHSA.In addition, this was undertaken to determine if rHSA could mitigatebackground current and increase analyte sensor current in rhNGAL-spikedurine. New NGAL sandwich ELISA reagents were created for this proof ofconcept in the i-STAT cartridge. This included using NGAL antibodiesproduced by Murine hybridoma cell lines 1-903-430 and 1-2322-455, whichwere each deposited with the American Type Culture Collection(hereinafter referred to as “ATCC”), 10801 University Blvd., Manassas,Va. 20110-2209, on Nov. 21, 2006. Cell line 1-903-430 was assigned ATCCAccession No. PTA-8026. Cell line 1-2322-455 was assigned ATCC AccessionNo. PTA-8024.

i-STAT immunosensors were printed with NGAL bead reagents and built intocartridges for testing. The cartridges were warmed to room temperaturebefore testing. Midstream urine from the same donor as in Experiments 1and 2 above was collected in a sterile tube (pH˜7.0) and endogenous NGALlevel was determined with an on-market NGAL ELISA Kit (BioPortoDiagnostics KIT037) in order to correct spiking values. For NGAL i-STATcartridge proof of concept testing, rhNGAL protein was sourced from theARCHITECT® (Abbott) rhNGAL calibrators. Urine was tested +/−rhNGAL and+/−rHSA (45 mg/mL). Whole blood was also tested with spiked amounts ofrhNGAL and signals were generated, revealing that the i-STAT systemcould measure NGAL in whole blood. rhNGAL was spiked into urine samplesat final concentrations of 0.5, 2 and 10 ng/mL. Spiked samples weremixed on a nutator (Clay Adams, Parsippany, N.J.) at room temperaturefor 10 minutes. NGAL antibody conjugate was spiked into the test samplesto a final concentration of 1.2 μg/mL just prior to cartridge loading. Afinal volume of 20 μL spiked sample was added to the cartridge inlet tocarry out the sandwich ELISA replicates.

FIG. 12 is chronoamperometric data for NGAL in urine with and withoutexogenous albumin on the i-STAT platform. A linear relationship ofamperometric signal and corresponding rhNGAL spikes was observed for lowlevels of rhNGAL (up to 10 ng/mL); this was the first evidence thatrhNGAL can be measured using i-STAT cartridge technology/methodologies.Occasionally, there was a loss in conductivity between thereference/analyte/conductivity bar sensors (vertical oval in FIG. 12)leading to an interruption in signal generation during analysis. Thiswas more pronounced at lower rhNGAL spike levels indicating that sensorwetup and maintenance was being compromised, likely due to low inherentmatrix protein levels. This was supported by the finding that samplesfortified with rHSA were able to avoid breaks in signal generation. Buteven with adjustment of reagent parameters, signal saturation began ataround 100 ng/mL NGAL (FIG. 13), which indicates that not even the fullnormal range can be measured for this analyte using the current i-STATparadigm. This is in contrast to the ARCHITECT uNGAL (urine NGAL)platform which covers a range from 10-6000 ng/mL (sandwich immunoassaywith paramagnetic particles, autodilution and wash functions).

rhNGAL was measured in urine on the i-STAT platform using a traditionalsandwich ELISA approach. Interruption of amperometric signal generationin low-level rhNGAL spike samples was mitigated by the introduction ofexogenous rHSA (˜45 mg/mL). The current immunoassay sandwich paradigm onthe i-STAT system is insufficient to cover the dynamic range of NGAL inurine. A modification to the platform to include the capability forcompetitive immunoassay formats was tested next.

Example 4 (Competitive Immunoassay for NGAL in Buffer on Microplates)

Off cartridge testing was employed to ascertain the tracer concentrationneeded in the i-STAT cartridge to effectively measure across the NGALdynamic range described above using a competitive ELISA format. rhNGALwas spiked at various concentrations into a TB S/1% BSA, pH 8 solution(0.05 M Tris buffered saline NaCl—0.138 M; KCl—0.0027 M). Typicalmethodology was used to perform a plate ELISA. To coat the microtitreplate with capture antibody: 50 μL/well of 5 μg/mL capture antibody(LF68057) in bicarbonate buffer was left to incubate for 3 hours at roomtemperature. The wells were then washed with PBS several times beforeadding 1% BSA in TBS to block the wells for 2 hours at room temperature.TBS, pH 8.0 was used to wash the wells after blocking and the plate wasblotted to remove excess moisture. 25 μL rhNGAL dilutions were mixedwith an equal volume of ALP-NGAL tracer such that the final amount oftracer per test condition covered 1, 2, 4, 8, 16, 32, 64, 96, 128, 160,and 192 ng (final tracer diluted rhNGAL concs tested with each of thetracer amounts were 0, 1, 50, 100, 1500, 6000, 12000 ng/mL). Incubationproceeded at room temperature in a covered plate for 1 hour. Wells werewashed 3 times with TBS, pH 8.0 then blotted dry; TMB substrate wasprepared (Sigma Blue AP Substrate A plus B). 100 μL substrate was addedto each well and colour development was allowed to take place.Measurements at 595 nm were taken and values were corrected for blank.Log NGAL concentration versus OD595 is plotted in FIG. 14. Good slope(sensitivity) was observed in the normal plasma range (40-110 ng/mL) andbeyond. Tracer range between 32-128 ng per test seemed acceptable tocover the NGAL dynamic range; competitive ELISA using this tracer rangewith LF68057 capture antibody was tried next in urine on the i-STATcartridge.

Example 5 (Competitive Immunoassay for NGAL in Urine on the i-STATCartridge)

10 mM phosphate buffered saline (PBS) containing 0.01% Tween 20 (pH 7.2)was used as the assay buffer. The wash buffer was a 10 mM PBS containing0.05% Tween 20 (pH 7.2). Using the LF68057 capture and HSA referencebeads formulated as in the preferred embodiment below, sensors wereprinted and built into NGAL i-STAT cartridges for testing. rhNGAL(sourced from the ARCHITECT (Abbott) rhNGAL calibrators) was spiked intodonor urine along with 45 mg/mL final concentration of rHSA and varioustracer amounts (to test the range 32-128 ng per test urine sample;prepared as in Example 4). 20 μL of amended samples were loaded intocartridges for the competitive immunoassay test cycle (mixing andanalysis conditions were kept similar to sandwich ELISA settings in thei-STAT; time to complex formation and conditions for wash and analysiscycle should be sufficient for both the sandwich and competitive formatswith NGAL and ALP label). A tracer level of about 100 ng per 20 μL urinesample combined with the typical amount of capture antibody on thei-STAT bead preparations (˜0.2 μg/mL with respect to sample volume;varying this allows for shifts in assay sensitivity) was found to besensitive enough over the NGAL range to be used in the competitive assayon the i-STAT; no dilution of high concentration samples was necessaryfor the competitive format.

rhNGAL can be measured in urine by a competitive immunoassay formatusing the i-STAT system. Capture NGAL antibody linked to polystyrenebeads, an alkaline phosphatase NGAL tracer, exogenous rHSA and anappropriate buffering solution were used in conjunction with the typicali-STAT cartridge shell to create a working urine NGAL competitiveimmunoassay which covers the appropriate NGAL range (FIG. 15).

Many types of immunoassay devices and processes have been describedherein and the following jointly owned patents and applications areincorporated by reference for further understanding of these devices andprocesses. A disposable sensing device for successfully measuringanalytes in a sample of blood is disclosed in U.S. Pat. No. 5,096,669.It employs a reading apparatus and a cartridge that fits into thereading apparatus for the purpose of measuring analyte concentrations ina sample of blood. Additionally, U.S. Pat. No. 7,723,099 describes animmunoassay device with an immuno-reference electrode; U.S. Pat. No.7,682,833 describes an immunoassay device with improved sample closure;U.S. Patent Application Publication No. 2004/0018577 describes amultiple hybrid immunoassay; U.S. Patent Application Publication No.2012/0295290 describes reducing interference from leukocytes in animmunoassay; U.S. Pat. No. 7,419,821 describes an apparatus and methodsfor analyte measurement and immunoassay; U.S. Pat. No. 8,084,272addresses ameliorating interferences from heterophile antibodies; U.S.Patent Application Publication No. 2010/0167301 describes a device andmethods for immunoassay using nucleotide conjugates; and U.S. PatentApplication Publication No. 2012/0034684 describes a magneticimmunosensor that may be applicable for use with a urine sample, each ofwhich is incorporated herein by reference in their entireties.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. It is intended thatthe scope of the present invention be limited solely by the scope of thefollowing claims. In addition, it should be appreciated by those skilledin the art that a plurality of the various embodiments of the invention,as described above, may be coupled with one another and incorporatedinto a single reader device.

We claim:
 1. A method for performing an immunoassay for a target analytein an amended urine sample, the method comprising: providing a testdevice comprising: a first region comprising reagents; and a secondregion comprising a sensor comprising at least one electrode coated witha capture molecule capable of binding to the target analyte to form animmunocomplex, wherein the reagents comprise: (i) a water solubleprotein for blocking non-specific binding of immunoglobulins at the atleast one electrode, (ii) a buffer, and (iii) a scavenger for reducingnon-specific current generation from electroactive species at the atleast one electrode; contacting a urine sample with the reagents in thefirst region to amend the urine sample such that a dissolvedconcentration of the water soluble protein within the amended urinesample is in a range of about 0.02 to 225 mg/mL; contacting the amendedurine sample with the second region to form the immunocomplex; anddetermining a concentration of the target analyte within the amendedurine sample by detecting the immunocomplex using the at least oneelectrode.
 2. The method of claim 1, wherein: the water soluble proteinis recombinant or non-recombinant human serum albumin, porcine serumalbumin, castor bean albumin, salmon serum albumin, bovine serumalbumin, or a mixture thereof.
 3. The method of claim 1, furthercomprising amending the urine sample with urease to reduce a ureaconcentration of the urine sample below a preselected urea threshold. 4.The method of claim 1, wherein the target analyte is neutrophilgelatinase-associated lipocalin (NGAL).
 5. The method of claim 1,wherein: the buffer adjusts a pH of the urine sample to within apreselected range; and the buffer is selected from the group consistingof: glycine, 3-(N-morpholino) propanesulfonic acid (MOPS),tris(hydroxymethyl)aminomethane (Tris), tricine, acetate, borate,2-(N-morpholino)ethanesulfonic acid (MES), and2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES).
 6. The method of claim 1, further comprising amending the urinesample with glutamine synthetase or any other urea cycle enzyme toconsume ammonium.
 7. The method of claim 1, further comprising amendingthe urine sample with a sequestering enzyme to reduce and sequesterexcess phosphate below a preselected phosphate threshold.
 8. The methodof claim 7, wherein the sequestering enzyme is adenylate kinase.
 9. Amethod for performing an immunoassay for a target analyte in an amendedurine sample, the method comprising: introducing an undiluted orpartially diluted urine sample into a sample chamber of a cartridgethrough a sample entry port; amending the urine sample with reagentssuch that a dissolved concentration of water soluble protein within theamended urine sample is in a range of about 0.02 to 225 mg/mL, whereinthe reagents comprise: (i) a water soluble protein for blockingnon-specific binding of immunoglobulins at a sensor, (ii) a buffer,(iii) a conjugate molecule labeled with an enzyme capable of binding tothe target analyte, and (iv) a scavenger for reducing non-specificcurrent generation from electroactive species at at least electrode;contacting the amended urine sample with a sensor on the cartridge,wherein the sensor comprises a capture molecule and the at least oneelectrode, the a capture molecule is capable of binding to the targetanalyte bound to the conjugate molecule labeled with the enzyme to forman immunocomplex, and the at least one electrode is configured to detectthe immunocomplex; and determining a concentration of the target analytewithin the amended urine sample by detecting the immunocomplex using theat least one electrode.
 10. The method of claim 9, wherein: the watersoluble protein is recombinant or non-recombinant human serum albumin,porcine serum albumin, castor bean albumin, salmon serum albumin, bovineserum albumin, or a mixture thereof.
 11. The method of claim 9, furthercomprising amending the urine sample with urease to reduce a ureaconcentration of the urine sample below a preselected urea threshold.12. The method of claim 9, wherein the target analyte is neutrophilgelatinase-associated lipocalin (NGAL).
 13. The method of claim 9,wherein: the buffer adjusts a pH of the urine sample to within apreselected range; and the buffer is selected from the group consistingof: glycine, 3-(N-morpholino) propanesulfonic acid (MOPS),tris(hydroxymethyl)aminomethane (Tris), tricine, acetate, borate,2-(N-morpholino)ethanesulfonic acid (MES), and2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES).
 14. The method of claim 9, further comprising amending the urinesample with glutamine synthetase or any other urea cycle enzyme toconsume ammonium.
 15. The method of claim 9, further comprising amendingthe urine sample with a sequestering enzyme to reduce and sequesterexcess phosphate below a preselected phosphate threshold.
 16. The methodof claim 15, wherein the sequestering enzyme is adenylate kinase.